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Rapid Determination of Protein Solubility and Stability Conditions for NMR Studies Using Incomplete Factorial Design

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Abstract

Sample preparation constitutes a crucial and limiting step in structural studies of proteins by NMR. The determination of the solubility and stability (SAS) conditions of biomolecules at millimolar concentrations stays today empirical and hence time- and material-consuming. Only few studies have been recently done in this field and they have highlighted the interest of using crystallogenesis tools to optimise sample conditions. In this study, we have adapted a method based on incomplete factorial design and making use of crystallisation plates to quantify the influence of physico-chemical parameters such as buffer pH and salts on protein SAS. A description of the experimental set up and an evaluation of the method are given by case studies on two functional domains from the bacterial regulatory protein LicT as well as two other proteins. Using this method, we could rapidly determine optimised conditions for extracting soluble proteins from bacterial cells and for preparing purified protein samples sufficiently concentrated and stable for NMR characterisation. The drastic reduction in the time and number of experiments required for searching protein SAS conditions makes this method particularly well-adapted for a systematic investigation on a large range of physico-chemical parameters.

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Abbreviations

BME:

beta-mercaptoethanol

DTT:

dithiothreitol

EDTA:

ethylenediamine tetraacetic acid

FFD:

full factorial design

IFD:

incomplete factorial design

IPTG:

isopropyl-beta-d-thiogalactopyranoside

LB:

Luria Bertani

SAS:

solubility and stability

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TRIS:

tris-(hydroxymethyl) aminomethane.

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Authors and Affiliations

  1. A contribution from the Centre de Biochimie Structurale, CNRS UMR 5048/INSERM UMR 554/Universito de Montpellier 1, 29 rue de Navacelles, 34090, Montpellier Cedex, France

    Thierry Ducat, Nathalie Declerck, Thierry Gostan, Michel Kochoyan & Hélène Déméné

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  1. Thierry Ducat
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  2. Nathalie Declerck
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  3. Thierry Gostan
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  4. Michel Kochoyan
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  5. Hélène Déméné
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Correspondence to Hélène Déméné.

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Ducat, T., Declerck, N., Gostan, T. et al. Rapid Determination of Protein Solubility and Stability Conditions for NMR Studies Using Incomplete Factorial Design. J Biomol NMR 34, 137–151 (2006). https://doi.org/10.1007/s10858-006-0003-0

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